Replication defective adenoviruses are being considered as vectors in therapeutic applications of gene therapy, as well as research tools in studying gene function. Important to their successful utilization is the development of techniques to isolate new recombinants quickly, which are not contaminated with wild-type virus. We describe a modification of the traditional technique to create recombinant adenoviruses in which a 5' plasmid containing vector sequence is cotransfected into 293 cells with viral DNA. In our protocol, the viral DNA is derived from the 3' portion of an E1-deleted recombinant that expresses the green fluorescent protein. Visualization of the cotransfection by fluorescent microscopy distinguishes recombinant plaques (nonfluorescent or 'white plaque') from background plaques (green fluorescent or 'green plaque'). Using this approach we have been able to increase substantially the success and throughput for creating new recombinants while minimizing contamination. This has been used to isolate adenoviral vectors deleted in a number of essential genes.